Investigating the role of Kinesin family in lung adenocarcinoma via integrated bioinformatics approach

Lung cancer is the leading cause of mortality from cancer worldwide. Lung adenocarcinoma (LUAD) is a type of non-small cell lung cancer (NSCLC) with highest prevalence. Kinesins a class of motor proteins are shown to be involved in carcinogenesis. We conducted expression, stage plot and survival analyses on kinesin superfamily (KIF) and scrutinized the key prognostic kinesins. Genomic alterations of these kinesins were studied thereafter via cBioPortal. A protein–protein interaction network (PPIN) of selected kinesins and 50 closest altering genes was constructed followed by gene ontology (GO) term and pathway enrichment analyses. Multivariate survival analysis based on CpG methylation of selected kinesins was performed. Lastly, we conducted tumor immune infiltration analysis. Our results found KIF11/15/18B/20A/2C/4A/C1 to be significantly upregulated and correlated with poor survival in LUAD patients. These genes also showed to be highly associated with cell cycle. Out of our seven selected kinesins, KIFC1 showed the highest genomic alteration with highest number of CpG methylation. Also, CpG island (CGI) cg24827036 was discovered to be linked to LUAD prognosis. Therefore, we deduced that reducing the expression of KIFC1 could be a feasible treatment strategy and that it can be a wonderful individual prognostic biomarker. CGI cg24827036 can also be used as a therapy site in addition to being a great prognostic biomarker.

www.nature.com/scientificreports/ employed, therapeutic resistance to these treatments is a primary cause of treatment failure. Understanding the underlying molecular pathways of carcinogenesis is thus critical for developing effective LC therapies. Human kinesin superfamily members (KIFs) consist of 14 kinesin family members, kinesin-1 to kinesin-14, according to the standardized nomenclature adopted by the kinesin research group 11 . There are 45 members in the KIFs superfamily, including 39 N-kinesins, three M-kinesins, and three C-kinesins 12 . KIF proteins are a family of motor proteins that move molecules and depend on microtubules. They have ATPase activity as well as motion characteristics. They bind to microtubules and then move along the microtubules, carrying protein complexes, organelles, and messenger RNAs (mRNAs) [12][13][14] . In recent years, it has come to light that several KIFs contribute uniquely to the process of mitosis, also known as cell division, by taking part in the motion of chromosomes and spindles 15,16 . Additionally, individual kinesins are also essential for a number of other cellular processes, such as endocytosis and transcytosis, intracellular transport 14 .
Mitosis, the process by which eukaryotic cells divide, creates two daughter cells with approximately equal amounts of the cell's nucleus, cytoplasm, organelles, and membrane. It is possible that mistakes in this process could lead to the death of cell, abnormalities (including deletion of gene, translocation of chromosome, or the duplication of chromosomes), and even cancer. Since mitosis is so intricately controlled therefore, any alteration or changes in KIF expression or function could potentially cause cancer. Kinesins and motor proteins with abnormal expression are crucial mitotic process regulators and potential targets in human malignancies [17][18][19] . Human cancer is a genetic disorder characterized by uncontrolled cell development, hence inhibiting kinesins may provide a unique approach to managing this disease.
Thus, identifying anomalous kinesin gene expression could be utilized as a biomarker for early tumor diagnosis and targeting kinesins could also be a novel approach for cancer therapy. Therefore, in the current study, we conducted a comprehensive bioinformatics analysis to identify the key kinesins influencing the prognosis of LUAD cancer patients. We performed expression and stage plot analyses of KIFs across the cancer genome atlas (TCGA)-LUAD patient samples and reported only significant ones. Next, we proceeded with overall survival (OS) analysis followed by mutational, enrichment, and protein-protein interaction network (PPIN) analyses. At last, we obtained KIFC1 as final prognostic biomarker responsible for LUAD pathogenesis. KIFC1 can be further used for early detection of LUAD patients and targeted therapy or personalized medicine.

Materials and methods
Kinesins expression and stage analysis across LUAD cohort. Gene expression profiling interactive analysis v2 (GEPIA 2) web-based tool 20 (http:// gepia2. cancer-pku. cn/) was accessed for comparing the relative mRNA expression level of all kinesin family members across TCGA-LUAD cohort and matched TCGA normal and GTEx data. The expression values from GEPIA were already transformed into log 2 (TPM + 1) values followed by differential analysis. Pathological stage plot analysis was also done with GEPIA 2 to investigate the kinesin family members' expression with respect to different pathological stages in LUAD. The threshold used in GEPIA for mRNA expression level comparison across LUAD and normal samples were as follows: pvalue < 0.05 and log 2 (foldchange) > 1 . Kinesins statistically significant in both expression and stage plot analyses were selected for further analyses.
Prognostic analysis of kinesins across LUAD cohort. Kaplan-Meier (KM) plotter 21,22 (https:// kmplot. com/ analy sis/) was queried for prognostic analysis of kinesins having significance in expression and stage plot analyses. We generated KM plots of only those kinesins which showed significant OS across LUAD patient samples. The microarray LUAD patients were bifurcated into higher and lower expression groups based on their median values. The redundant samples were removed in the quality control section, and biased arrays were excluded. Hazard ratio (HR) with the corresponding 95% confidence interval (CI), logrank p value and median survival were calculated. logrank p value < 0.05 was considered as a statistically significant threshold for assessing the prognosis of kinesins between two expression groups.
Validation of prognostic kinesins using cBioPortal. We queried the cBioPortal for Cancer Genomics 23 (https:// www. cbiop ortal. org/) for investigating the mutations and putative copy number alterations (CNAs) of prognostically significant kinesins. The LUAD dataset (TCGA, Firehose Legacy) was chosen to perform our analysis.
Validation of prognostic kinesins using GEO and correlation analysis. We queried the NCBI-GEO 24 (https:// www. ncbi. nlm. nih. gov/ geo/) using "LUAD" and "Lung Adenocarcinoma" as suitable keywords for extracting LUAD-associated mRNA expression profile. All the search results were further trimmed down in accordance with the following inclusion criteria: (1) the samples present in dataset(s) must belong to 'Homo Sapiens'; (2) dataset(s) type must be 'expression profiling by array'; (3) both preprocessed and raw files of the dataset(s) must be available; (4) the dataset(s) submission date to GEO must be within last 10 years (i.e. 2012-2022); (5) the dataset(s) must be comprising both tumor and healthy control tissue samples; (6) the dataset(s) must comprise at least 25 samples. Any abstracts, case reports, review-based articles, cell-line-based experimental study designs, and studies devoid of healthy controls or non-human samples were excluded. Sequential steps of batch correction, probe ID to gene mapping, and duplicacy removal were performed as discussed previously 25 . The DEGs were screened corresponding to a Benjamini-Hochberg (BH)-p value < 0.0 and log 2 fold change > 0.5 utilizing limma 26 . The presence of key prognostic kinesins was checked in the DEGs list. Next, we accessed GEPIA 2 to perform pairwise correlation analysis of key prognostic kinesins across TCGA-LUAD and normal patients. p value < 0.05 was considered as the cutoff for statistical significance. Tumor infiltration analysis. We looked into the relationship between mRNA expression levels of prognostically significant kinesins with tumor-infiltrating immune cells such as B cells, CD8 + T cell, macrophage, and neutrophils across TCGA-LUAD patients using TIMER 2.0 33 (http:// timer. cistr ome. org/). To assess the statistical significance, Spearman correlation was used.
Methylation analysis. Prognostic analysis of single CpG methylation of selected genes of kinesin family in LUAD patients was conducted using MethSurv 34 (https:// biit. cs. ut. ee/ meths urv), a web tool for multivariate survival analysis based on CpG methylation data.

Prognostic analysis based on single CpG methylation of selected kinesins in LUAD patients.
We obtained the heatmaps of DNA methylation of selected kinesins using MethSurv. Among which cg04344917 CpG island (CGI) of KIF11, cg09053247 CGI of KIF15, cg01838385 CGI of KIF18B, cg07632946 CGI of KIF20A, cg20487572 CGI of KIF2C, cg27286863 CGI of KIF4A, cg2390442 CGI of KIFC1 showed the highest methylation levels (Fig. 8). Furthermore, we studied KM plots which revealed that cg24827036 CGI of KIFC1 were significantly associated with survival of LUAD patients (Fig. 9). A total of 461 patients were split into higher and lower expression groups. Higher methylated expression of KIFC1 worsened the OS of LUAD patients. www.nature.com/scientificreports/

Discussion
LUAD's malignancy results in high morbidity and fatality rate 35,36 . Despite advances in surgery, radiation, and chemotherapy, which have improved tumor patients' clinical prognosis and survival 37 , LUAD is still hard to www.nature.com/scientificreports/ treat because scientists don't fully understand the molecular mechanisms and basic signaling pathways in how LC works. It is expected that molecule-targeted therapy will be a revolutionary treatment technique for solid tumors, however, its efficacy and advantages remain restricted 38,39 . Because of chemoresistance and recurrence, the currently available therapeutic choices are limited. Therefore, a new and effective molecular target must be identified to cure LUAD. Members of the KIF gene family are mostly found in eukaryotic cells, namely microtubules. Experiments conducted in vitro have shown that the transport of proteins occurs in only one direction, along the microtubule's negative pole and in the direction of the positive pole. Therefore, the genes that make up the KIF family are responsible for controlling the movement of mass proteins both inside of cells and outside of cells. This control encompasses a variety of functions, such as moving organelles and vesicles that contain material and taking part in the process of cell mitosis 15,40,41 . There have been reports that several genes in the kinesin family are linked to different kinds of cancer [42][43][44] . KIF family member genes have been demonstrated in various cancer types to establish their prognostic and diagnostic capacities. In our current study, we performed expression analysis of kinesin family in LUAD which revealed overexpression of KIF11/12/15/23/18B/20A/2C/4A/C1 in tumor samples whereas KIF17/26A/1C were underexpressed in LUAD. Furthermore, we also studied mRNA expression based on cancer stage which showed overexpression of KIF11/15/23/18B/20A/2C/4A/C1 in tumor tissues. Furthermore, we evaluated the prognostic value of selected kinesins in LUAD patients. Our results showed that an increased KIF11/15/18B/20A/2C/4A/C1 expression is associated with poor OS in LUAD patients. So, by targeting these kinesins and decreasing their effect can be of therapeutic importance and patients' survival can be increased.
Our findings corroborate with multiple previous findings that showed overexpression of KIF11, KIF15, KIF18B, KIF20A, KIF2C, and KIF4A in LUAD tissues and when LUAD patients have high expression of these KIFs, their chances of survival are lower 38,45-49 . Next, we studied the genomic alterations of key kinesins which www.nature.com/scientificreports/ showed the highest alteration in KIFC1 (2.3%) as amplification being the most prominent type of alteration. Following that we constructed a PPIN of key kinesins and top 50 frequently altered genes and performed enrichment analysis. Our results showed high enrichment of kinesins in cell cycle and oocyte meiosis pathway, in biological processes named microtubule cytoskeleton organization involved in mitosis and mitotic spindle organization, microtubule binding and microtubule motor activity molecular functions and spindle and microtubule cytoskeleton cellular components. For cells to divide and multiply, they go through a series of events known as the cell cycle, and abnormalities in the control of the genes involved in the cell cycle have been linked to the development of tumours. Mutations in upstream signal transduction pathways or genetic abnormalities within genes that encode cell cycle proteins cause cancer 50 . Our result showed high enrichment of kinesins in cell cycle processes hence kinesins are involved in controlling these processes somewhat and regulating LUAD.
The other two typically active mechanisms in LC were oocyte meiosis and progesterone-mediated oocyte maturation. One cycle of DNA replication in meiosis is followed by two cycles of chromosomal segregation (Meiosis I and Meiosis II). Normally, oocytes are stopped during the G2 stage of meiosis I. Progesterone exposure releases www.nature.com/scientificreports/ them from this natural lock, allowing the two meiotic division cycles to resume and the oocyte to mature 51,52 . So, it makes sense that dysregulations in oocyte maturation and meiosis would impact the cell cycle process, and further cell cycle changes would impact normal bodily functions, increasing the likelihood that one would develop cancer. So, cell cycle, progesterone-mediated oocyte maturation, and oocyte meiosis play prominent roles in the progression of LUAD 53 and these two processes come under top 10 in the pathway enrichment analysis we did in our study showing the importance of kinesins in controlling these pathways in LUAD Microtubules are α -and β-tubulin heterodimers polymers. They exhibit highly dynamic behaviour, continuously engaging through processes of polymerization and de-polymerization, as well as lengthening and shortening. The fundamental components of the cytoskeleton are actin, intermediate filaments, and microtubules. They are crucial for various cell processes, including mitosis, the movement of vesicles and organelles inside cells, cell signaling, migration through cilia and flagella, cell shape and morphology 54 . So it can be said that any alteration in the function of kinesin from normal can impact these important pathways involved in LUAD progression. We also performed tumor immune infiltration analysis on our key kinesins as tumor-infiltrating immune cells are critical components of the tumour microenvironment (TME), influencing tumor growth and survival depending upon their type and interaction LC clearly displays an invasion of a wide variety of immune cell types comprising neutrophils, natural killer (NK) cells, macrophages, dendritic cells, T cells & B cells 55 . These cells perform multiple purposes and combine or oppose one another, producing the LC TME. Neutrophils make up   56 . Our data revealed the highest correlation of our key kinesins with infiltration abundances of neutrophils in LUAD patients. CD8 + T cell, also known as cytotoxic T lymphocytes, play a crucial role in mounting an efficient antitumor response. These cells can identify specific tumor-associated antigens (TAA) that are presented on major histocompatibility complex (MHC) class I molecules on the surface of cancer cells. Furthermore, they possess the ability to eliminate cancer cells directly 57 .
Our study found that infiltrations of CD8 + T cell and neutrophil correlated most with KIF15 expression levels. Also, B cell and macrophage infiltrations correlated most with KIF11 and KIF4A expression levels. Cancer is often caused by the inactivation of several tumor-suppressor genes through point mutations and deletion of chromosomes 58 . Recent research has shown that epigenetic changes are key to cancer development. Many genes have CGIs in their promoter regions, and abnormal methylation of these sites in cancer leads to transcriptional suppression. Epigenetic alterations are passed down through cell division, resulting in gene activity change but no changes in the sequence of DNA 59,60 . Changes in DNA methylation patterns are a key feature of many types of cancer, including LC.
So further we conducted a single CpG methylation-based prognostic analysis on key kinesins which showed that CpG methylation in KIFC1 was associated with poor prognosis in LUAD patients. KIFC1 is believed to be an oncogene in various types of cancers as it plays a crucial role in clustering multiple centrosomes to sustain tumor survival 61,62 .

Conclusions
Our research revealed a significant function for the kinesin family in initiating and progressing LUAD. KIF11/15/18B/20A/2C/4A/C1 mRNA expression levels were significantly upregulated and correlated with poor OS across LUAD patients. They were highly associated with cell cycle. Our results revealed the highest genomic alteration in KIFC1 with highest number of CpG methylation. cg24827036 CGI of KIFC1 was associated with poor prognosis across LUAD. We concluded that KIFC1 can be a great individual prognostic biomarker, and that inhibiting its expression could be a potential therapeutic approach. Additionally, CpG island cg24827036 can serve as a great prognostic biomarker and treatment site.

Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.  www.nature.com/scientificreports/